Efficient synthesis, biological evaluation, and docking study of isatin based derivatives as caspase inhibitors

Article abstract of DOI:10.1080/14756366.2020.1809388

ABTRACT: In this paper, a new series of isatin-sulphonamide based derivatives were designed, synthesised and evaluated as caspase inhibitors. The compounds containing 1-(pyrrolidinyl)sulphonyl and 2-(phenoxymethyl)pyrrolidin-1-yl)sulphonyl substitution at C5 position of isatin core exhibited better results compared to unsubstituted derivatives. According to the results of caspase inhibitory activity, compound 20d showed moderate inhibitory activity against caspase-3 and ?7 in?vitro compared to Ac-DEVD-CHO (IC₅₀ = 0.016 ± 0.002 μM). Among the studied compounds, some active inhibitors with IC_50s in the range of 2.33–116.91 μM were identified. The activity of compound 20d was rationalised by the molecular modelling studies exhibiting the additional van der Waals interaction of N-phenylacetamide substitution along with efficacious T-shaped π-π and pi-cation interactions. The introduction of compound 20d with good caspase inhibitory activity will help researchers to find more potent agents.

Full text of DOI:10.1080/14756366.2020.1809388

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Efficient synthesis, biological evaluation, and
docking study of isatin based derivatives as
caspase inhibitors
Loghman Firoozpour , Lixin Gao , Setareh Moghimi , Parvin Pasalar , Jamshid
Davoodi , Ming-Wei Wang , Zahra Rezaei , Armin Dadgar , Hoda Yahyavi ,
Massoud Amanlou & Alireza Foroumadi
To cite this article: Loghman Firoozpour , Lixin Gao , Setareh Moghimi , Parvin Pasalar , Jamshid
Davoodi , Ming-Wei Wang , Zahra Rezaei , Armin Dadgar , Hoda Yahyavi , Massoud Amanlou
& Alireza Foroumadi (2020) Efficient synthesis, biological evaluation, and docking study of isatin
based derivatives as caspase inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry,
35:1, 1674-1684, DOI: 10.1080/14756366.2020.1809388
To link to this article: https://doi.org/10.1080/14756366.2020.1809388
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1674–1684
https://doi.org/10.1080/14756366.2020.1809388
RESEARCH PAPER
Efficient synthesis, biological evaluation, and docking study of isatin based
derivatives as caspase inhibitors
Loghman Firoozpoura, Lixin Gaob, Setareh Moghimia, Parvin Pasalarc, Jamshid Davoodid, Ming-Wei Wangb,
Zahra Rezaeie, Armin Dadgarf, Hoda Yahyavia, Massoud Amanloue
and Alireza Foroumadia,e
aDrug Design and Development Research Center, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran,
b
c
Iran; National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China; Department of
d
Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran; Institute of Biochemistry and Biophysics, University of
e
Tehran, Tehran, Iran; Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran;
fPharmaceutical Sciences Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
ABTRACT
ARTICLE HISTORY
Received 27 August 2019
Revised 6 August 2020
Accepted 7 August 2020
In this paper, a new series of isatin-sulphonamide based derivatives were designed, synthesised and eval-
uated as caspase inhibitors. The compounds containing 1-(pyrrolidinyl)sulphonyl and 2-(phenoxymethyl)-
pyrrolidin-1-yl)sulphonyl substitution at C5 position of isatin core exhibited better results compared to
unsubstituted derivatives. According to the results of caspase inhibitory activity, compound 20d showed
KEYWORDS
moderate inhibitory activity against caspase-3 and ꢀ7 in vitro compared to Ac-DEVD-CHO (IC50
¼
Caspase inhibitor; Isatin
sulphonamides; docking
studies;
0.016 0.002 lM). Among the studied compounds, some active inhibitors with IC50s in the range of
2.33–116.91 lM were identified. The activity of compound 20d was rationalised by the molecular model-
ling studies exhibiting the additional van der Waals interaction of N-phenylacetamide substitution along
with efficacious T-shaped p-p and pi-cation interactions. The introduction of compound 20d with good
caspase inhibitory activity will help researchers to find more potent agents.
Pharmacophore; apoptosis
Introduction
respectively8–10. This knowledge along with the importance of this
family clearly helps medicinal chemists to design new specific
Caspases, cysteinyl aspartate-specific proteases, are a family of sig-
nalling molecules playing a key role in apoptosis. Apoptosis is a
physiological suicide process which gives an opportunity to dis-
mantle unwanted cells population during animal development
and tissue homeostasis1. Morphological changes such as DNA
strand breaks along with nuclear membrane damage occur as a
result of some biochemical events during apoptosis2. Two intrinsic
and extrinsic pathways are responsible for initiating the apoptosis
inhibitors of caspase enzymes11–19
.
Isatin sulphonamides are introduced as a new class of potent
and selective non-peptide caspase-3 and ꢀ7 inhibitors. Previously,
various isatin sulphonamide derivatives were prepared and eval-
uated as caspase-3 inhibitors20–25. The studies indicated the con-
nection between carbonyl group of isatin ring and cysteine thiol
in the binding site of the enzyme. 5-Pyrrolidinyl sulphonyl isatins
process. Binding of certain protein to the death receptor activates are evidently found effective in inhibition of the caspase-3 and ꢀ7
caspase-8 and subsequently triggers apoptosis by promoting
effector caspases (–3, ꢀ6, ꢀ7). It should be noted that caspase
enzymes are classified as initiator (caspase-2, caspase-8, caspase-9,
and caspase-10) and effector (caspase-3, caspase-6, and caspase-7)
in vitro. The selectivity of 5-pyrrolidinyl sulphonyl isatins is referred
to the interaction of pyrrolidine ring with S2 subsite of enzyme
without the interaction with S1 subsite of caspase-326. The side-
chains, attached to pyrrolidine meaning methoxymethyl or phe-
noxymethyl groups, occupy the S3 pocket. In this regard, many
studies have been focussed on the synthesis of several modified
isatin derivatives (1), relying on the structure-activity relationship
(SAR) studies (Figure 1). Interestingly, it was observed that good
IC50 values in nanomolar ranges are obtained when hydrophobic
groups are attached to the N-1 position of structure 1 (Figure 1).
Furthermore, the amide moiety is also found necessary in produc-
ing various potent inhibitors27–29. Considering the above men-
tioned findings about the importance of isatin sulphonamide
derivatives, especially as caspase-3 inhibitors and following our
ongoing projects on the design and synthesis of biologically
which are exploited in re sponse to proapoptotic signals3,4
.
Caspase-3, activated by the upstream caspase-8 and caspase-9, is
considered as a crucial mediator of apoptotic cell death in mam-
mals by which more than 500 cellular substrates are cleaved to
execute the apoptosis programme5,6. Regarding the close relation-
ship between apoptosis and the wide range of disease, caspase
inhibitors are capable of opening new paths to treat several dis-
eases involving immunodeficiency, Alzheimer’s, Parkinson’s,
Huntington’s diseases, ischaemia, brain trauma, and amyotrophic
lateral sclerosis7. Taking the obtained data from the X-ray struc-
ture of caspase-3 into account, four main binding sites (S1–S4) are
determined in which the binding to the S2 and S3-pockets are
responsible for inhibitory activity and selectivity of caspase-3, active agents30–37
,
we synthesised isatin based compounds
CONTACT Alireza Foroumadi
aforoumadi@yahoo.com; Loghman Firoozpour
firoozpour@gmail.com
Drug Design and Development Research Center, The
Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1675
1
2
3
IC50 =120 nM
IC50 = 44 nM
IC50 = 2.5 nM
IC50 = 46.7µM
IC50 = 0.086 µM
IC50 = 0.031 µM
Figure 1. Some of the reported caspase-3 inhibitors. IC50 ¼ 120 nM; IC50 ¼ 44 nM; IC50 ¼ 2.5 nM. IC50 ¼ 46.7mM; IC50 ¼ 0.086 mM; IC50 ¼ 0.031 mM
containing N-aryl acetamide and N-prop-2-yn-1-yl as caspase-3 decoupled 13 C-NMR spectrum of compounds showed characteris-
and ꢀ7 inhibitors through the structural modification of com-
tic signals at related aliphatic and aromatic regions which are in
agreement with the proposed structure.
pound 1.
Results and discussion
Biological activity
Chemistry
The inhibitory activities of newly synthesised 2–(2,3-dioxoindolin-
1-yl)-N-substituted phenyl acetamide, 1-(prop-2-yn-1-yl)indoline-
2,3-dione and two series of compounds containing 1-(pyrrolidinyl)-
sulphonyl and 2-(phenoxymethyl)pyrrolidin-1-yl)sulphonyl substitu-
tion at C5 position of isatin core (B, C, Table 1) against caspase-3
and ꢀ7 were evaluated by using the acetyl-DEVD-AMC fluorogenic
substrate assay. The results are expressed as inhibition percentage
and IC50 values in Table 1. We used Ac-DEVD-CHO as the posi-
tive control.
As can be seen in Table 1, those compounds containing no
substituent at C5 position of isatin core (R2 ¼ H) are weak inhibi-
tors compared to the positive control. All amounts are provided
as inhibition percentage at 20 lg/ml. Among this series, the best
and weakest activity was observed in 11c and 11f with inhibition
percentage of 71% and 5%, respectively. The presence of 2-(phe-
noxymethyl)pyrrolidine functionality on isatin core led to the
more active compounds against caspase-3 and ꢀ7 than that of
substituted ones with pyrrolidin-1-yl sulphonyl moiety.
In compounds 20a–k, the comparison between the para sub-
stituted derivatives revealed that the electron-donating substitu-
ents (20j, 20k) exhibited the lowest enzymatic inhibition. The
most active compound was the 4-chlorophenylacetamide contain-
ing derivative, meaning 20d against caspase-3 and ꢀ7.
Compounds 20a–c and 20f have also appreciable IC50 values and
can be regarded as moderated caspase-3 and ꢀ7 inhibitors in
comparison to Ac-DEVD-CHO (IC50 ¼ 0.016 0.002 lM). In com-
pounds 19a–k and 20 a–k, the least electronegative and more
First of all, the N-alkylated isatin derivatives (11a–k) were obtained
in 60–85% yields from the alkylation reaction of isatin 10 with
propargyl bromide or 2-chloro-N-arylacetamide derivatives38–40
,
synthesised from the reaction of chloroacetyl choride and aro-
matic amines (Part A, Scheme 1)41.
The synthesis of N-alkylated substituted 5-[1-(pyrrolidinyl)sul-
phonyl] isatin derivatives was started from heating isatin 10 in
chlorosulfonic acid at 60 ꢁC which is followed by amination with
pyrrolidine or 2-phenoxymethyl pyrrolidine in dimethyl formamide
(DMF)42. The subsequent hydrolysis in acetic acid and addition of
2-chloro-N-arylacetamides 9 or propargyl bromide led to com-
pounds 19a–k (64–85%) and 20a–k (47–65%) in good yields (Part
B, Scheme 1).
In this paper, 33 compounds are synthesised and their struc-
tures are deduced by IR, 1H, 13 C NMR, mass spectroscopy, and
elemental analysis. For example, the IR spectrum of these three
series showed the stretching bands, related to C¼O bonds of
ketone and amide functional groups at nearly 1700 and
1670 cmꢀ1, respectively. The mass spectrum of each compound
displayed the molecular ion (Mþ) peak, which is consistent with a
1:1 adduct, formed by the substitution at NH of isatin and loss of
chlorine and bromine atom of propargyl bromide or 2-chloro-N-
arylacetamide derivatives. The 1H-NMR spectrum of compounds
exhibited the characteristic signals at d 4.3–4.6 and 8.2–8.8 ppm
related to NCH2 and NH, respectively. The characteristic signals
related to pyrrolidine and isatin moiety at aliphatic and aromatic
region confirmed the structures of final compounds. The 1H- bulky atom, bromine, had clear negative effect on inhibitory

1676
L. FIROOZPOUR ET AL.
(A)
(B)
Scheme 1. (A) Synthesis route for A series. Reagents and conditions. a: CH2Cl2, Et3N; b: NaH, DMF (B) Synthesis route for B series. Reagents and conditions. a: ClSO3H;
b: pyrrolidine or 16, Et3N, DMF, c: acetic acid; d: 9 or propargyl bromide, NaH, DMF, 0 ˚C; e: p-toluenesulfonyl chloride, pyridine; f: phenol, NaH, THF; g: TFA, CH2Cl2.
potency of the compound compared to fluorine and chlorine con- substitution provided enough length for more efficient interac-
taining derivatives. As previously reported, compounds with a tions, like an additional van der Waals interaction between LeuA
selectivity index greater than 1.5 are considered as selective inhib- 168 and ThrA 166 and phenyl moiety. Table 2, presentesd the
itors of caspase-3, so, compounds 19a, 19d, 19e, 20c, 20d, and comparision between the type of interaction and involved amino
20e exhibited this selectivity. Regarding the significant activity acid residues of the most active compound, 20d, and isatin sul-
and selectivity of compound 20d, this compound could be phonamide. These interactions along with distances are schemat-
studied for further modification to develop novel hit compounds.
ically presented in Figure 3. Superimposition of the binding pose
of 20d and natural ligand at the 1GFW active site is shown in
Figure 4. The binding interaction energy of compound 20d is
ꢀ4.04 kcal/mol, which stated that this compound is less potent
than statin sulphonamide (-5.44 kcal/mol) towards caspase-3.
Docking study
To investigate the binding mode of these potent inhibitors,
molecular docking computations were performed using Autodock
Tools (ver.1.5.6) programme43. Compound 20d was docked into
the active site of caspase-3 crystallographic structure (PDB ID:
Conclusion
1GFW), retrieved from protein data bank (http://www.rcsb.org/ A series of novel isatin-sulphonamide derivatives were designed,
pdb/home/home.do) (Figure 2). The phenyl ring of phenoxymethyl synthesised and evaluated for their caspase-3 and ꢀ7 inhibitory
group formed pi-cation interaction with HIS:121. The isatin core activity. The results showed that most of the synthesised com-
formed T-shaped p-p interactions with His 121 and Tyr 204. His pounds exhibited moderate inhibitory activity against caspase-3
121 formed a carbon hydrogen bond in isatin sulphonamide crys- and ꢀ7. The results revealed that 4-chloro phenylacetamide
tal ligand. A Pi-alkyl interaction is formed between the oxygen of derivative 20d exhibited the best profile of inhibitory activity on
sulphonyl group and Trp 206 and Tyr 204. The carbonyl moiety caspase-3 with IC50 value of 2.33 mM. The docking studies showed
interacted through p-sulfur with Cys 163 in compound 20d and the perfect binding of compound 20d to the active site of cas-
through p-hydrogen bond in isatin sulphonamide. The p-p stacked pase-3 enzyme. The prepared product 20d in the present study
interaction is formed between isatin core and Phe 256 in isatin may be subjected to further optimisation to find more effective
sulphonamide and compound 20d. Moreover, N-phenylacetamide agent as caspase-3 inhibitor.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1677
reaction was continued for 15 min at 0 ꢁC. Corresponding N-phe-
nylacetamides 9 or propargyl bromide (0.25 mmol) was added and
the reaction was continued for one hour. TLC was used to find
reaction completion time. Water (20 ml) was added to the reaction
mixture and extracted with ethyl acetate. Resulted crude product
was purified over flash column chromatography (mobile phase:
ethyl acetate: hexane 20:80) to yield pure products 11a–k.
1-(Prop-2-yn-1-yl)indoline-2,3-dione (11a)44: White solid;
Yield: 85%; m.p. 158–160 ꢁC; IR (KBr, cmꢀ1): 1718 (C¼OKetone), 1678
(C¼OAmide); 1H-NMR (500 MHz, DMSO-d6): 2.90 (s, 1H, CHAcetylene),
4.36 (s, 2H, CH2), 7.22 (d, J ¼ 8.5 Hz, 1H, H7), 7.35 (d, J ¼ 8.5 Hz, 1H),
7.47 (t, J ¼ 7.85 Hz, 1H), 7.52 (t, J ¼ 7.85 Hz, 1H); 13 C NMR
(125 MHz, DMSO-d6): 37.0, 75.1, 82.0, 126.2, 126.6, 128.3, 135.1,
135.1, 145.6, 150.1, 163.3, 181.1; Anal. Calcd. For C11H7NO2: C,
71.35; H, 3.81; N, 7.56; Found: C, 71.07; H, 3.58; N, 7.84.
2–(2,3-Dioxoindolin-1-yl)-N-phenylacetamide (11b): White
solid; Yield 78%; m.p. 174–176 ꢁC; IR (KBr, cmꢀ1): 3348 (NH), 1710
(C¼OKetone), 1680 (C¼OAmide), 1660 (C¼OAmide); 1H-NMR (500 MHz,
DMSO-d6): 4.64 (s, 2H, CH2), 7.07 (d, J ¼ 7.2 Hz, 1H), 7.14 (t,
J ¼ 7.2 Hz, 1H), 7.19 (t, J ¼ 7.9 Hz, 1H), 7.32 (t, J ¼ 7.9 Hz, 2H), 7.49
(d, J ¼ 7.9 Hz, 2H), 7.70–7.62 (m, 2H),7.94 (s, 1H, NH); 13 C-NMR
(125 MHz, DMSO-d6): 43.9, 110.2, 117.9, 121.2, 125.7, 127.6, 132.7,
134.8, 145.7, 148.5, 151.6, 162.6, 166.3, 182.1; Anal. Calcd. For
C16H12N2O3: C, 68.56; H, 4.32; N, 9.99; Found: C, 68.27; H, 4.04; N,
10.17; MS (m/z, %): 280 (Mþ, 41) 146 (100), 134 (25), 90 (57), 77
(33), 55 (76).
2–(2,3-Dioxoindolin-1-yl)-N-(4-fluorophenyl)acetamide (11c):
White solid; Yield 70%; m.p. 199–201 ꢁC; IR (KBr, cmꢀ1): 3340 (NH),
1700 (C¼OKetone), 1688 (C¼OAmide), 1665 (C¼OAmide); 1H-NMR
(500 MHz, DMSO-d6): 4.64 (s, 2H, CH2), 6.89 (d, J ¼ 7.5 Hz, 2H), 6.95
(d, J ¼ 7.0 Hz, 1H), 6.99 (d, J ¼ 7.5 Hz, 2H), 7.14 (t, J ¼ 7.0 Hz, 1H),
7.58–7.63 (m, 2H), 8.05 (s, 1H, NH); 13 C-NMR (125 MHz, DMSO-d6):
46.6, 110.6, 121.3, 125.6, 127.2, 128.3, 130.5, 134.5, 148.1, 150.9,
Figure 2. 2 D and 3 D representations of 20d interactions with caspase-3
active site.
158.3 (JC-F
¼
250 Hz), 162.6, 167.0, 181.8; Anal. Calcd. For
C16H11FN2O3: C, 64.43; H, 3.72; N, 9.39; Found: C, 64.67; H, 3.44; N,
9.55; MS (m/z, %): 298 (Mþ, 63) 146 (100), 152 (32), 96 (48),
57 (40).
Experimental
Chemistry
N-(4-Chlorophenyl)-2–(2,3-dioxoindolin-1-yl)acetamide (11d):
White solid; Yield: 72%; m.p. 177–179 ꢁC; IR (KBr, cmꢀ1): 3356 (NH),
1708 (C¼OKetone), 1682 (C¼OAmide), 1655 (C¼OAmide); 1H-NMR
(500 MHz, CDCl3): 4.38 (s, 2H, CH2), 6.99 (d, J ¼ 8.0 Hz, 1H), 7.21 (d,
J ¼ 6.9 Hz, 2H), 7.24 (t, J ¼ 8.0 Hz, 1H), 7.29 (d, J ¼ 6.9 Hz, 2H), 7.61
(t, J ¼ 8.0 Hz, 2H), 8.01 (s, 1H, NH). 13 C-NMR (125 MHz, CDCl3): 43.8,
110.9, 117.7, 124.5, 125.6, 128.7, 137.3 (2 C), 138.7, 150.1, 158.5,
165.7, 182.4. Anal. Calcd. For C16H11ClN2O3: C, 61.06; H, 3.52; N,
8.90; Found: C, 61.37; H, 3.74; N, 9.05; MS (m/z, %): 316 (M þ 2þ,
39), 314 (Mþ, 14) 168 (35), 146 (100), 152 (52), 112 (64), 90 (28),
56 (40).
N-(4-Bromophenyl)-2–(2,3-dioxoindolin-1-yl)acetamide (11e):
White solid; Yield: 66%; m.p. 179–181 ꢁC; IR (KBr, cmꢀ1): 3330 (NH),
1698 (C¼OKetone), 1670 (C¼OAmide), 1656 (C¼OAmide); 1H-NMR
(500 MHz, DMSO-d6): 4.49 (s, 2H, CH2), 6.90 (d, J ¼ 7.0 Hz, 1H), 6.90
(d, J ¼ 7.0 Hz, 1H), 7.08 (d, J ¼ 7.5 Hz, 2H), 7.16–7.21 (m, 1H),
7.54–7.56 (m, 2H), 7.64 (d, J ¼ 7.9 Hz, 2H), 8.00 (s, 1H, NH). 13 C-
NMR (125 MHz, DMSO-d6): 45.0, 110.6, 115.4, 117.2, 118.3, 124.9,
126.8, 135.8, 138.0, 149.8, 150.4, 158.3, 163.3, 182.1, Anal. Calcd.
For C16H11BrN2O3: C, 53.50; H, 3.09; N, 7.80; Found: C, 53.87; H,
3.44; N, 7.57; MS (m/z, %): 359 (M þ 2þ, 26), 357 (Mþ, 24), 211 (47),
154 (69), 146 (100), 90 (36), 56 (50).
5-[1-(Pyrrolidinyl)sulphonyl] isatin derivatives 18 were prepared by
the reaction of isatin 7, chlorosulfonic acid, pyrrolidine or 2-phe-
noxymethyl pyrrolidine 16 (Scheme 1). 2-Chloro-N-phenylaceta-
mide derivatives 938–40 and 2-phenoxymethyl pyrrolidine 1622
used in the synthesis of target products were conveniently pre-
pared based on the previously reported procedure.
Other starting materials, chemical reagents, and solvents used
in this study were commercially available (from Merck and Aldrich
Chemicals) and were used without further purification. TLC was
conducted on silica gel 250 micron. Melting points were deter-
mined on a Kofler hot stage apparatus and are uncorrected. The
IR spectra were run on a Shimadzu 470 spectrophotometer (potas-
sium bromide disks). Mass spectra were recorded on an Agilent
Technologies (HP) 5973 mass spectrometer operating at an ionisa-
tion potential of 70 eV. The NMR spectra were recorded on a
Varian unity 500 spectrometer, and the chemical shifts (d) are
reported in parts per million (ppm) relative to tetramethylsilane
(TMS) as an internal standard.
General procedure for the N-alkylation of isatin, 5-[1-(pyrrolidi-
nyl)sulphonyl] isatins, 5-((2-(phenoxymethyl)pyrrolidin-1-yl)sul-
phonyl)isatin
2–(2,3-Dioxoindolin-1-yl)-N-(2-nitrophenyl)acetamide
(11f):
White solid; Yield: 77%; m.p. 186–188 ꢁC; IR (KBr, cmꢀ1): 3340 (NH),
1725 (C¼O Ketone), 1685 (C¼O Amide), 1665 (C¼OAmide); 1H-NMR
Sodium hydride (0.25 mmol) was added to the stirred solution of
isatin 10 or intermediate 18 (0.25 mmole) in DMF (3 ml), and the (500 MHz, CDCl3): 4.58 (s, 2H, CH2), 7.00 (d, J ¼ 7.0 Hz, 1H), 7.11 (t,

1678
L. FIROOZPOUR ET AL.
Table 1. Structures of compounds 11a–k, 19a–k, and 20a–k displaying inhibitory effects on caspase-3 and -7.
Caspase-3
IC50
Caspase-7
IC50
Caspase-3
IC50
Caspase-7
IC50
SId
Caspase-3
IC50
Caspase-7
IC50
SId
R1
Entry
(μM) a
11
(μM) a
11
(μM) a
19
(μM) a
19
(μΜ) a
20
(μΜ) a
20
(33.21 7.13)d
N.D.
24.97 0.95
61.38 10.11
2.46
0.83
5.67 0.62
6.62 0.74
1.16
1.32
a
(65.0 3.22)
N.D.
23.97 0.91
20.05 4.65
3.98 0.41
5.25 0.14
b
(71.41 10.63)
N.D.
19.05 1.88
14.18 2.47
0.74
4.87 0.63
12.17 4.12
2.49
c
d
e
f
27.30 3.29
38.69 4.20
>20
102.48 6.94
116.91 13.01
27.40 5.14
13.11 4.37
3.75
3.02
1.62
2.05
1.42
0.60
2.33 0.33
7.15 0.47
3.98 0.45
3.77 1.69
14.70 1.84
5.67 0.41
(55.19 1.21)
(13.90 4.22)
N.D.
N.D.
(5.11 1.50)
N.D.
N.D.
<1.37
0.58
(27.77 6.12)
22.72 1.48
11.92 0.99
7.19 0.65
g
(26.70 1.05)
(11.01 4.45)
(53.61 3.27)
N.D.
N.D.
N.D.
24.45 4.33
39.70 2.84
36.50 3.47
14.53 3.27
21.31 2.78
36.50 5.15
0.59
0.54
1.04
8.25 1.22
7.17 0.58
32.63 4.33
3.72 0.71
6.13 1.65
30.36 6.21
0.45
0.85
0.93
h
i
j
(55.50 6.25)
N.D.
31.91 2.34
27.64 4.65
0.87
30.66 4.41
22.56 2.47
0.73
k
aIC50 values are expressed as Mean SD of three experiments. bN.D. ¼ Not determined. cIC50 amount for Ac-DEVD-CHO is 0.016 0.002 lM. dThe values given in
bracket are percentage inhibition. dSelectivity Index (SI) was calculated as IC50 caspase-7/IC50 caspase-3.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1679
J ¼ 7.0 Hz, 1H), 7.27–7.33 (m, 2H), 7.37 (d, J ¼ 8.0 Hz, 1H), 7.58 (t,
2–(2,3-Dioxoindolin-1-yl)-N-(3-nitrophenyl)acetamide (11g):
J ¼ 7.0 Hz, 1H), 7.80 (t, J ¼ 8.0 Hz, 1H), 7.92 (d, J ¼ 8.0 Hz, 1H), 8.50 White solid; Yield: 75%; m.p. 191–192 ꢁC; IR (KBr, cmꢀ1): 3348 (NH),
(s, 1H, NH). 13 C-NMR (125 MHz, CDCl3): 45.8, 123.6, 127.3, 129.4,
1725 (C¼OKetone), 1680 (C¼OAmide), 1658 (C¼OAmide); 1H-NMR
129.9, 129.9, 132.3, 134.8, 135.2, 136.4, 139.9, 145.6, 146.5, 151.0,
161.2, 168.4, 179.3. Anal. Calcd. For C16H11N3O5: C, 59.08; H, 3.41;
N, 12.92; Found: C, 59.37; H, 3.14; N, 13.17; MS (m/z, %): 325 (Mþ,
48), 179 (51), 146 (100), 123 (44), 92 (29), 57 (42).
(500 MHz, CDCl3): 4.49 (s, 2H, CH2), 7.08–7.11 (m, 1H), 7.24 (d,
J ¼ 8.0 Hz, 1H), 7.32–7.34 (m, 1H), 7.56–7.58 (m, 1H), 7.64 (d,
J ¼ 7.0 Hz, 1H), 7.83 (t, J ¼ 7.0 Hz, 1H), 8.00 (s, 1H), 8.18 (d,
J ¼ 7.5 Hz, 1H), 8.39 (s, 1H, NH). 13 C-NMR (125 MHz, CDCl3): 45.60,
124.1, 127.8, 127.9, 130.0, 130.2, 132.2, 135.4, 135.9, 139.4, 140.3,
146.0, 146.9, 160.4, 168.6, 179.6. Anal. Calcd. For C16H11N3O5: C,
59.08; H, 3.41; N, 12.92; Found: C, 59.35; H, 3.73; N, 13.19.
2–(2,3-Dioxoindolin-1-yl)-N-(4-nitrophenyl)acetamide (11h):
White solid; Yield: 69%; m.p. 179–181 ꢁC; IR (KBr, cmꢀ1): 3338 (NH),
1718 (C¼OKetone), 1680 (C¼O Amide), 1665 (C¼OAmide); 1H-NMR
(500 MHz, DMSO-d6): 4.49 (s, 2H, CH2), 7.13 (d, J ¼ 7.2 Hz, 1H),
7.19–7.24 (m, 1H), 7.38–7.41 (m, 2H), 7.66–7.69 (m, 1H), 7.85–7.88
(m, 1H), 8.00–8.04 (m, 2H), 8.37 (s, 1H, NH). 13 C-NMR (125 MHz,
DMSO-d6): 46.1, 124.6, 127.3, 128.1, 129.1, 131.5, 135.6, 139.1,
145.4, 145.7, 150.4, 161.6, 167.0, 178.4. Anal. Calcd. For
C16H11N3O5: C, 59.08; H, 3.41; N, 12.92; Found: C, 59.31; H, 3.79;
N, 13.11.
Table 2. The interactions of compound 20d and natural ligand in 1GFW at the
active site.
Interaction type
20d
Isatin Sulphonamide
Van der waals
Conventional hydrogen bond
Carbon hydrogen bond
Pi-pi stacked
Pi-pi T-shaped
Pi-alkyl
–
–
–
–
Arg 207, Gly 122
His 121
Phe 256
Tyr 204
Trp 206
–
Phe 256
His 121, Tyr 204
Trp 206. Tyr 204
His 121
Pi-cation
Pi-hydrogen bond
Pi-sulfur
Tyr 204
Cys 163
Cys 163
–
2–(2,3-Dioxoindolin-1-yl)-N-(4-(trifluoromethoxy)phenyl)ace-
tamide (11i): White solid; Yield: 75%; m.p. 209– 211 ꢁC; IR (KBr,
cmꢀ1): 3348 (NH), 1721 (C¼OKetone), 1680 (C¼OAmide), 1665
(C¼OAmide); 1H-NMR (500 MHz, DMSO-d6): 4.52 (s, 2H, CH2),
7.23–7.27 (m, 3H), 7.11 (d, J ¼ 7.5 Hz, 1H), 7.38 (d, J ¼ 8.5 Hz, 2H),
7.57 (t, J ¼ 7.5 Hz, 1H), 7.93 (d, J ¼ 7.5 Hz, 1H), 8.35 (s, 1H, NH). 13 C-
NMR (125 MHz, DMSO-d6): 45.2, 116.0, 121.6, 124.5, 126.5, 128.3,
128.6, 131.7, 134.2, 135.4, 138.9, 149.2, 161.9, 166.7, 178.9. Anal.
Calcd. For C17H11F3N2O4: C, 56.05; H, 3.04; N, 7.69; Found: C, 55.91;
H, 3.42; N, 7.84; MS (m/z, %): 364 (Mþ, 26), 218 (Mþ, 18), 162 (44),
146 (100), 90 (51), 56 (32).
2–(2,3-Dioxoindolin-1-yl)-N-(p-tolyl)acetamide (11j): White
solid; Yield: 60%; m.p. 196–198 ꢁC; IR (KBr, cmꢀ1): 3354 (NH), 1728
1
(C¼OKetone), 1688 (C¼O Amide), 1659 (C¼OAmide); H-NMR (500 MHz,
DMSO-d6): 2.34 (s, 3H), 4.59 (s, 2H, CH2), 6.97 (d, J ¼ 7.6 Hz, 1H),
7.21 (d, J ¼ 8.0 Hz, 2H), 7.27 (d, J ¼ 7.6 Hz, 1H), 7.41 (d, J ¼ 8.0 Hz,
2H), 7.71 (d, J ¼ 7.8 Hz, 1H), 7.88 (t, J ¼ 7.8 Hz, 1H), 8.37 (s, 1H, NH).
Anal. Calcd. For C17H14N2O3: C, 69.38; H, 4.79; N, 9.52; Found: C,
Figure 3. Superimposition of the binding pose for 20d and natural ligand at the
1GFW active site.
Figure 4. 2 D representations of 20d (A) and isatin sulphonamide (B) interactions with caspase-3 active site.

1680
L. FIROOZPOUR ET AL.
69.51; H, 4.92; N, 9.66; MS (m/z, %): 294 (Mþ, 42), 148 (34), 146 290–292 ꢁC; IR (KBr, cmꢀ1): 3334 (NH), 1705 (C¼OKetone), 1675
(100), 91 (44), 56 (68).
(C¼OAmide), 1660 (C¼OAmide); 1H-NMR (500 MHz, DMSO-d6):
1.62–1.66 (m, 4H, CH2-Pyrrole) 3.00–3.07 (m, 4H, CH2-Pyrrole), 4.49 (s,
2H, CH2), 7.11 (d, J ¼ 8.6 Hz, 1H), 7.49 (d, J ¼ 8.5 Hz, 2H), 7.61 (d,
2–(2,3-Dioxoindolin-1-yl)-N-(4-methoxyphenyl)acetamide
(11k): White solid; Yield: 68%; m.p. 201–203 ꢁC; IR (KBr, cmꢀ1):
3354 (NH), 1728(C¼OKetone), 1688 (C¼OAmide), 1659 (C¼OAmide); J ¼ 8.5 Hz, 2H), 7.78 (d, J ¼ 7.5 Hz, 1H), 8.18 (s, 1H), 8.52 (s, 1H, NH);
1H-NMR (500 MHz, DMSO-d6): 3.70 (s, 3H, OMe), 4.47 (s, 2H, CH2), 13 C-NMR (125 MHz, DMSO-d6): 20.7, 43.8, 62.7, 113.3, 123.2, 125.9,
6.88 (d, J ¼ 8.0 Hz, 2H), 7.08–7.11 (m, 1H), 7.20–7.25 (m, 3H),
126.0, 127.0, 127.4, 128.5, 129.1, 130.7142.5, 160.8, 165.5, 181.8;
7.55–7.57 (m, 1H), 7.91–7.92 (m, 1H), 8.22 (s, 1H, NH). 13 C-NMR Anal. Calcd. For C20H18BrN3O5S: C, 48.79; H, 3.69; N, 8.53; Found:
(125 MHz, DMSO-d6): 45.9, 55.0, 113.8, 116.1, 121.6, 124.4, 126.5, C, 48.45; H, 3.46; N, 8.78; MS (m/z, %): 493 (M þ 2þ, 28), 491 (Mþ,
128.4, 134.2, 149.0, 149.2, 161.9 (2 C), 167.6, 178.4. Anal. Calcd. For
C17H14N2O4: C, 65.80; H, 4.55; N, 9.03; Found: C, 65.55; H, 4.28; N,
26), 278 (51), 211 (29), 154 (48), 146 (100), 133 (24).
2–(2,3-Dioxo-5-(pyrrolidin-1-ylsulfonyl)indolin-1-yl)-N-(2-nitro-
9.33; MS (m/z, %): 310 (Mþ, 26), 164 (18), 108 (44), 146 (100), 92 phenyl)acetamide (19f): White solid; Yield: 74%; m.p. 195–197 ꢁC;
(51), 58 (32).
IR (KBr, cmꢀ1): 3345 (NH), 1725 (C¼OKetone), 1685 (C¼OAmide), 1660
1-(Prop-2-yn-1-yl)-5-(pyrrolidin-1-ylsulfonyl)indoline-2,3-dione (C¼OAmide); 1H-NMR (500 MHz, DMSO-d6): 1.61–1.66 (m, 4H, CH2-
(19a): White solid; Yield: 82%; m.p. 203–205 ꢁC; IR (KBr, cmꢀ1): Pyrrole), 2.99–3.07 (m, 4H, CH2-Pyrrole), 4.49 (s, 2H, CH2), 7.12 (d,
3345 (NH), 1710 (C¼OKetone), 1680 (C¼OAmide), 1660 (C¼OAmide); J ¼ 7.0 Hz, 1H), 7.24 (t, J ¼ 8.0 Hz, 1H), 7.52 (d, J ¼ 8.6 Hz, 1H), 7.73
1H-NMR (500 MHz, DMSO-d6): 1.63–1.70 (m, 4H, CH2-Pyrrole), (t, J ¼ 8.0 Hz, 1H), 8.01 (s, 1H), 8.12 (t, J ¼ 7.0 Hz, 1H), 8.37 (d,
2.98–3.04 (m, 4H, CH2-Pyrrole), 3.24 (s, 1H, CH), 4.73 (s, 2H, CH2), J ¼ 7.0 Hz, 1H), 8.66 (s, 1H, NH); Anal. Calcd. For C20H18N4O7S: C,
7.13 (d, J ¼ 8.5 Hz, 1H), 7.82 (d, J ¼ 8.5 Hz, 1H), 8.08 (s, 1H); 13 C- 52.40; H, 3.96; N, 12.22; Found: C, 52.53; H, 4.09; N, 12.35; MS (m/z,
NMR (125 MHz, DMSO-d6): 25.9, 47.2, 61.9, 71.9, 73.8, 112.0, 126.5, %): 458 (Mþ, 41), 279 (37), 211 (40), 163 (27), 146 (100), 135 (19).
128.0, 136.6, 152.5, 161.5, 166.6, 179.8; Anal. Calcd. For
2–(2,3-Dioxo-5-(pyrrolidin-1-ylsulfonyl)indolin-1-yl)-N-(3-nitro-
C15H14N2O4S: C, 56.59; H, 4.43; N, 8.80; Found: C, 56.77; H, 4.11; N, phenyl)acetamide (19g): White solid; Yield 73%; m.p. 223–225 ꢁC;
8.57; MS (m/z, %): 318 (Mþ, 42), 279 (26), 208 (51), 146 (100), IR (KBr, cmꢀ1): 3340 (NH), 1720 (C¼OKetone), 1680 (C¼OAmide), 1660
71 (22).
(C¼OAmide); 1H-NMR (500 MHz, DMSO-d6): 1.58–1.65 (m, 4H, CH2-
Pyrrole), 2.89–3.03 (m, 4H, CH2-Pyrrole), 4.38 (s, 2H, CH2), 7.12 (d,
2–(2,3-Dioxo-5-(pyrrolidin-1-ylsulfonyl)indolin-1-yl)-N-phenyl-
acetamide (19b): White solid; Yield: 85%; m.p. 238–240 ꢁC; IR (KBr, J ¼ 7.5 Hz, 1H), 7.32 (t, J ¼ 7.5 Hz, 1H), 7.71 (d, J ¼ 7.5 Hz, 1H), 7.82
cmꢀ1): 3364 (NH), 1725 (C¼OKetone), 1684 (C¼OAmide), 1655 (d, J ¼ 7.5 Hz, 1H), 8.01 (s, 1H), 8.17 (s, 1H), 8.20 (s, 1H), 8.54 (s, 1H,
(C¼OAmide); 1H-NMR (500 MHz, DMSO-d6): 1.60– 1.67 (m, 4H, CH2- NH); 13 C-NMR (125 MHz, DMSO-d6): 25.1, 49.5, 61.3, 110.1, 122.8,
Pyrrole), 3.08–3.12 (m, 4H, CH2-Pyrrole), 4.49 (s, 2H, CH2), 6.92 (d, 123.4, 126.1, 128.2, 129.0, 130.4, 132.1, 134.1, 135.0, 143.4, 150.9,
J ¼ 7.5 Hz, 1H), 7.12 (d, J ¼ 8.8 Hz, 2H), 7.36 (d, J ¼ 8.0 Hz, 1H), 7.44 158.9, 167.6, 180.0; Anal. Calcd. For C20H18N4O7S: C, 52.40; H, 3.96;
(t, J ¼ 7.5 Hz, 1H), 7.64 (t, J ¼ 8.0 Hz, 2H), 7.96 (s, 1H), 8.48 (s, 1H, N, 12.22; Found: C, 52.78; H, 4.14; N, 11.90.
NH); 13 C-NMR (125 MHz, DMSO-d6): 23.8, 47.8, 62.2, 123.7, 129.0,
2–(2,3-Dioxo-5-(pyrrolidin-1-ylsulfonyl)indolin-1-yl)-N-(4-nitro-
129.2, 131.5, 134.4, 142.6, 143.2, 143.4, 145.4, 147.2, 162.3, 167.2, phenyl)acetamide (19h): White solid; Yield: 69%; m.p. 216–218 ꢁC;
182.3; Anal. Calcd. For C20H19N3O5S: C, 58.10; H, 4.63; N, 10.16; IR (KBr, cmꢀ1): 3340 (NH), 1728 (C¼OKetone), 1680 (C¼OAmide), 1646
Found: C, 58.37; H, 4.91; N, 10.35; MS (m/z, %): 413 (Mþ, 39), 279 (C¼OAmide); 1H-NMR (500 MHz, DMSO-d6): 1.64–1.67 (m, 4H, CH2-
(43), 146 (100), 134 (57), 71 (29).
Pyrrole), 2.94–2.98 (m, 4H, CH2-Pyrrole), 4.68 (s, 2H, CH2), 7.12 (d,
J ¼ 7.5 Hz, 1H), 7.46 (dd, J ¼ 8.0, 3.5 Hz, 2H), 7.68 (d, J ¼ 7.5 Hz,
2–(2,3-Dioxo-5-(pyrrolidin-1-ylsulfonyl)indolin-1-yl)-N-(4-fluo-
rophenyl)acetamide (19c): White solid; Yield: 80%; m.p. 1H), 7.90 (s, 1H), 8.21 (dd, J ¼ 8.0 Hz, J ¼ 3.5 Hz, 2H), 8.51 (s, 1H,
191–193 ꢁC; IR (KBr, cmꢀ1): 3345 (NH), 1711 (C¼OKetone), 1670 NH); 13 C-NMR (125 MHz, DMSO-d6): 25.2, 48.1, 60.3, 111.3, 122.9,
(C¼OAmide), 1654 (C¼OAmide); 1H-NMR (500 MHz, DMSO-d6): 123.4, 130.4, 132.1, 133.2, 134.1, 142.4, 143.3, 149.2, 160.1, 167.6,
1.69–1.77 (m, 4H, CH2-Pyrrole) 2.98–3.11 (m, 4H, CH2-Pyrrole), 4.41 (s, 183.4; Anal. Calcd. For C20H18N4O7S: C, 52.40; H, 3.96; N, 12.22;
2H, CH2), 4.41 (s, 2H, CH2), 6.94 (d, J ¼ 7.5 Hz, 1H), 7.14 (d, Found: C, 52.67; H, 4.23; N, 12.53.
J ¼ 8.8 Hz, 2H), 7.36 (d, J ¼ 8.8 Hz, 2H), 7.42 (d, J ¼ 7.5 Hz, 1H), 8.05
2–(2,3-Dioxo-5-(pyrrolidin-1-ylsulfonyl)indolin-1-yl)-N-(4-(tri-
(s, 1H), 8.49 (s, 1H, NH); 13 C-NMR (125 MHz, DMSO-d6): 25.5, 48.0, fluoromethoxy)phenyl)acetamide (19i): White solid; Yield: 64%;
60.0, 110.5, 114.4 (JC-F ¼ 6.75 Hz), 119.2, 126.8 (JC-F ¼ 24.5 Hz), m.p. 266–267 ꢁC; IR (KBr, cmꢀ1): 3340 (NH), 1710 (C¼OKetone), 1678
128.1, 131.0, 135.6, 149.0, 151.6, 158.6, 162.0 (JC–F ¼ 245 Hz), (C¼OAmide), 1656 (C¼OAmide); 1H-NMR (500 MHz, DMSO-d6):
165.6, 183.2; Anal. Calcd. For C20H18FN3O5S: C, 55.68; H, 4.21; N, 1.65–1.70 (m, 4H, CH2-Pyrrole), 3.08–3.13- (m, 4H, CH2-Pyrrole), 4.66 (s,
9.74; Found: C, 55.37; H, 4.51; N, 9.49; MS (m/z, %): 431 (Mþ, 27), 2H, CH2), 6.85 (d, J ¼ 7.0 Hz, 2H), 7.14 (d, J ¼ 8.5 Hz, 1H), 7.20 (d,
278 (39), 154 (51), 146 (100), 135 (57), 95 (31).
J ¼ 7.0 Hz, 2H), 7.44 (d, J ¼ 8.5 Hz, 1H), 8.04 (s, 1H), 8.54 (s, 1H, NH);
N-(4-Chlorophenyl)-2–(2,3-dioxo-5-(pyrrolidin-1-ylsulfonyl)in-
13 C-NMR (125 MHz, DMSO-d6): 25.8, 47.4, 62.5, 113.1, 123.8, 129.0,
dolin-1-yl)acetamide (19d): White solid; Yield: 71%; m.p. 129.2, 131.5, 134.4, 142.6, 143.2, 143.4, 147.2, 151.4, 160.2, 167.3,
300–302 ꢁC; IR (KBr, cmꢀ1): 3330 (NH), 1706 (C¼OKetone), 1685 180.9; Anal. Calcd. For C21H18F3N3O6S: C, 50.70; H, 3.65; N, 8.45;
(C¼OAmide), 1658 (C¼OAmide); 1H-NMR (500 MHz, DMSO-d6): Found: C, 50.47; H, 3.81; N, 8.12; MS (m/z, %): 497 (Mþ, 51), 279
1.65–1.58 (m, 4H, CH2-Pyrrole). 2.98–3.05 (m, 4H, CH2-Pyrrole), 4.24 (s, (41), 218 (23), 161 (29), 146 (100), 77 (22).
2H, CH2), 6.77 (d, J ¼ 8.65 Hz, 1H), 7.38 (d, J ¼ 8.0 Hz, 2H), 7.65 (d,
J ¼ 8.0 Hz, 2H), 7.72 (d, J ¼ 8.65 Hz, 1H), 8.08 (s, 1H), 8.34 (s, 1H,
NH); 13 C-NMR (125 MHz, DMSO-d6): 26.7, 47.5, 62.8, 111.2, 120.3,
2–(2,3-Dioxo-5-(pyrrolidin-1-ylsulfonyl)indolin-1-yl)-N-(p-toly-
l)acetamide (19j): White solid; Yield: 77%; m.p. 248–250 ꢁC; IR
(KBr, cmꢀ1): 3356 (NH), 1716 (C¼OKetone), 1688 (C¼OAmide), 1653
125.9, 126.8, 128.4, 129.3, 129.6, 134.9, 144.5, 151.4, 158.4, 165.9, (C¼OAmide); 1H-NMR (500 MHz, DMSO-d6): 1.64–1.69 (m, 4H, CH2-
Pyrrole), 2.25 (s, 3H, CH3), 3.09–3.17 (m, 4H, CH2-Pyrrole), 4.64 (s, 2H,
CH2), 7.12 (d, J ¼ 7.85 Hz, 2H), 7.39 (d, J ¼ 8.25 Hz, 1H), 7.43 (d,
J ¼ 7.85 Hz, 2H), 7.86 (s, 1H), 8.11 (d, J ¼ 8.25 Hz, 1H), 8.47 (s, 1H,
NH); 13 C-NMR (125 MHz, DMSO-d6):18.3, 24.9, 49.4, 59.9, 112.0,
182.8; Anal. Calcd. For C20H18ClN3O5S: C, 53.63; H, 4.05; N, 9.38;
Found: C, 53.85; H, 3.78; N, 9.09; MS (m/z, %): 449 (M þ 2þ, 36),
447 (Mþ, 11), 276 (43), 168 (35), 146 (100), 134 (18), 110 (29).
N-(4-Bromophenyl)-2–(2,3-dioxo-5-(pyrrolidin-1-ylsulfonyl)in-
dolin-1-yl)acetamide (19e): White solid; Yield: 68%; m.p. 119.2, 126.9, 127.5, 128.9, 129.4, 130.6, 131.6, 134.3, 148.7, 158.2,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1681
165.1, 183.3; Anal. Calcd. For C21H21N3O5S: C, 59.00; H, 4.95; N, 4.18 (d, J ¼ 13.0 Hz, 1H, O-CH2-Diastropic), 4.58 (s, 2H, N-CH2), 7.03 (t,
9.83; Found: C, 59.37; H, 5.21; N, 10.12; MS (m/z, %): 427 (Mþ, 52), J ¼ 7.0 Hz, 2H), 7.34 (d, J ¼ 7.5 Hz, 1H), 7.36–7.41 (m, 3H), 7.48–7.55
278 (41), 150 (29), 146 (100), 134 (39), 57 (36).
(m, 4H), 8.03 (d, J ¼ 8.0 Hz, 1H), 8.07 (s, 1H),8.58 (s, 1H, NH); 13 C-
2–(2,3-Dioxo-5-(pyrrolidin-1-ylsulfonyl)indolin-1-yl)-N-(4-
NMR (125 MHz, DMSO-d6): 22.5, 28.6, 46.6, 28.6, 62.4, 117.3, 119.2,
methoxyphenyl)acetamide (19k): White solid; Yield: 73%; m.p. 127.2, 128.3, 129.0, 129.2, 130.8, 131.0, 132.8, 134.2, 135.2, 135.7,
194–196 ꢁC; IR (KBr, cmꢀ1): 3356 (NH), 1716 (C¼OKetone), 1688
139.9, 152.0, 161.7, 166.5, 180.8; Anal. Calcd. For C27H24ClN3O6S: C,
(C¼OAmide), 1653 (C¼OAmide); 1H-NMR (500 MHz, DMSO-d6): 58.53; H, 4.37; N, 7.58; Found: C, 58.73; H, 4.18; N, 7.29; MS (m/z,
1.64–1.68 (m, 4H, CH2-Pyrrole), 2.82 (t, J ¼ 6.0 Hz, 4H, CH2-Pyrrole), 3.94 %): 554 (Mþ, 26), 385 (62), 240 (48), 168 (21), 146 (100), 112 (17),
(s, 3H, O-CH3), 4.19 (s, 2H, CH2), 6.85 (d, J ¼ 8.0 Hz, 2H), 7.10 (d,
77 (41), 58 (55).
J ¼ 8.5 Hz, 1H), 7.41 (d, J ¼ 8.0 Hz, 2H), 7.50 (d, J ¼ 8.5 Hz, 1H), 8.12
(S)-N-(4-Bromophenyl)-2–(2,3-dioxo-5-((2-(phenoxymethyl)-
(s, 1H), 8.46 (s, 1H, NH);13C-NMR (125 MHz, DMSO-d6): 25.1, 45.7, pyrrolidin-1-yl)sulphonyl)indolin-1-yl)acetamide (20e): White
55.3, 63.7, 110.3, 114.5, 122.4 (2 C), 123.4, 126.5, 129.7, 132.1, solid; Yield: 51%; m.p. 196–198 ꢁC; IR (KBr, cmꢀ1): 1722
134.0, 159.1, 160.2, 167.6, 181.1; Anal. Calcd. For C21H21N3O6S: C, (C¼OKetone), 1680 (C¼OAmide), 1660 (C¼OAmide); 1H-NMR (500 MHz,
56.88; H, 4.77; N, 9.48; Found: C, 56.57; H, 4.31; N, 9.12; MS (m/z, DMSO-d6): 1.78–1.83 (m, 4H, CH2-Pyrrole), 2.60–2.63 (m, 2H, CH2-
%): 443 (Mþ, 52), 278 (41), 150 (29), 146 (100), 134 (39), 57 (36).
Pyrrole), 3.40–3.45 (m, 1H, CHChiral), 4.13 (d, J ¼ 13.0 Hz, 1H, O-CH2-
(S)-5-((2-(Phenoxymethyl)pyrrolidin-1-yl)sulphonyl)-1-(prop-2-
Diastropic), 4.28 (d, J ¼ 13.0 Hz, 1H, O-CH2-Diastropic), 4.68 (s, 2H, N-
yn-1-yl)indoline-2,3-dione (20a): White solid; Yield: 65%; m.p. CH2), 7.03 (t, J ¼ 7.0 Hz, 2H), 7.16–7.21 (m, 3H), 7.37–7.42 (m, 5H),
282–284 ꢁC; IR (KBr, cmꢀ1): 1725 (C¼OKetone), 1680 (C¼OAmide), 8.02 (d, J ¼ 8.0 Hz, 1H), 8.14 (s, 1H), 8.56 (s, 1H, NH); 13 C-NMR
1658 (C¼OAmide); 1H-NMR (500 MHz, DMSO-d6): 1.61–1.67 (m, 4H, (125 MHz, DMSO-d6): 22.6, 28.6, 46.6, 58.1, 62.4, 116.0, 117.5, 117.7,
CH2-Pyrrole), 2.66–2.69 (m, 2H, CH2-Pyrrole), 3.38–3.42 (m, 2H, 119.0, 127.2, 128.7, 129.0, 129.6, 130.9, 135.2, 135.7, 139.0, 139.8,
CHAcetylene,CHPyrrole), 4.00–4.09 (m, 2H, O-CH2), 4.61 (s, 2H, N-CH2), 150.0, 162.3, 167.8, 179.0; Anal. Calcd. For C27H24BrN3O6S: C, 54.19;
6.93 (d, J ¼ 7.2 Hz, 2H), 7.28 (t, J ¼ 7.2 Hz, 3H), 7.42 (d, J ¼ 7.7 Hz, H, 4.04; N, 7.02; Found: C, 54.34; H, 4.44; N, 7.38; MS (m/z, %): 600
1H), 7.87 (s, 1H), 8.21 (d, J ¼ 7.7 Hz, 1H); 13 C-NMR (125 MHz, (M þ 2þ, 37), 598 (Mþ, 35), 385 (41), 240 (29), 155 (42), 146 (100),
DMSO-d6): 23.6, 26.0, 46.1, 58.5, 62.4, 72.0, 73.6, 118.6, 123.7, 77 (36).
124.4, 126.2, 128.5, 129.8, 138.5, 139.5, 151.9, 161.9, 164.0, 179.6;
(S)-2–(2,3-Dioxo-5-((2-(phenoxymethyl)pyrrolidin-1-yl)sulpho-
Anal. Calcd. For C22H20N2O5S: C, 62.25; H, 4.75; N, 6.60; Found: C, nyl)indolin-1-yl)-N-(2-nitrophenyl)acetamide (20f): White solid;
62.51; H, 4.97; N, 6.32; MS (m/z, %): 424 (Mþ, 48), 385 (31), 248 Yield: 47%; m.p. 180–182 ꢁC; IR (KBr, cmꢀ1): 1720 (C¼OKetone), 1685
(54), 208 (40), 176 (23), 146 (100), 107 (21), 77 (30).
(C¼OAmide), 1654 (C¼OAmide); 1H-NMR (500 MHz, DMSO-d6):
1.79–1.83 (m, 4H, CH2-Pyrrole), 2.63–2.67 (m, 2H, CH2-Pyrrole),
(S)-2–(2,3-Dioxo-5-((2-(phenoxymethyl)pyrrolidin-1-yl)sulpho-
nyl)indolin-1-yl)-N-phenylacetamide (20b): White solid; Yield: 3.38–3.41 (m, 1H, CHChiral), 3.86 (d, J ¼ 13.0 Hz, 1H, O-CH2-Diastropic),
56%; m.p. 238–240 ꢁC; IR (KBr, cmꢀ1): 1720 (C¼OKetone), 1688 4.17 (d, J ¼ 13.0 Hz, 1H, O-CH2-Diastropic), 4.68 (s, 2H, N-CH2),
(C¼OAmide), 1660 (C¼OAmide); 1H-NMR (500 MHz, DMSO-d6): 6.99–7.04 (m, 3H), 7.28 (t, J ¼ 8.0 Hz, 1H), 7.34 (t, J ¼ 7.0 Hz, 2H),
1.78–1.84 (m, 4H, CH2-Pyrrole), 2.62–2.65 (m, 2H, CH2-Pyrrole), 7.38–7.41 (m, 2H), 7.77 (t, J ¼ 8.0 Hz, 1H), 8.03 (d, J ¼ 8.0 Hz, 1H),
3.30–3.36 (m, 1H, CHChiral), 3.87 (d, J ¼ 12.0 Hz, 1H, O-CH2-Diastropic), 8.07 (d, J ¼ 7.5 Hz, 1H), 8.18 (s, 1H), 8.62 (s, 1H, NH); 13 C-NMR
4.14 (d, J ¼ 12.0 Hz, 1H, O-CH2-Diastropic), 4.60 (s, 2H, N-CH2), (125 MHz, DMSO-d6): 22.6, 28.6, 46.6, 58.1, 62.4, 116.0, 117.5, 117.9,
6.99–7.05 (m, 3H), 7.37–7.43 (m, 6H), 7.50 (d, J ¼ 8.0 Hz, 2H), 8.02 119.2, 120.9, 127.2, 128.3, 130.1, 131.4, 131.6, 135.2, 135.8, 140.0,
(d, J ¼ 8.0 Hz, 1H), 8.10 (s, 1H), 8.46 (s, 1H, NH); 13 C-NMR (125 MHz, 142.4, 143.6, 144.0, 161.1, 167.0, 179.3; Anal. Calcd. For
DMSO-d6): 22.5, 27.6, 45.6, 58.5, 63.2, 117.7, 119.2, 120.9, 127.2, C27H24N4O8S: C, 57.44; H, 4.28; N, 9.92; Found: C, 57.74; H, 4.49; N,
128.4, 128.7, 128.9, 129.2, 129.5, 134.0, 135.2, 135.7, 139.8, 151.1, 10.21; MS (m/z, %): 564 (Mþ, 48), 324 (39), 385 (53), 240 (27), 179
161.3, 166.2, 179.7; Anal. Calcd. For C27H25N3O6S: C, 62.42; H, 4.85; (33), 144 (100), 123 (61), 77 (25).
N, 8.09; Found: C, 62.66; H, 4.97; N, 8.39; MS (m/z, %): 519 (Mþ,
52), 278 (41), 150 (29), 146 (100), 134 (39), 77 (36).
(S)-2–(2,3-Dioxo-5-((2-(phenoxymethyl)pyrrolidin-1-yl)sulpho-
nyl)indolin-1-yl)-N-(3-nitrophenyl)acetamide (20g): White solid;
Yield: 52%; m.p. 181–183 ꢁC; IR (KBr, cmꢀ1): 1725 (C¼OKetone), 1686
(S)-2–(2,3-Dioxo-5-((2-(phenoxymethyl)pyrrolidin-1-yl)sulpho-
nyl)indolin-1-yl)-N-(4-fluorophenyl)acetamide (20c): White solid; (C¼OAmide), 1656 (C¼OAmide); 1H-NMR (500 MHz, DMSO-d6):
Yield: 53%; m.p. 108–110 ꢁC; IR (KBr, cmꢀ1): 1725 (C¼OKetone), 1690 1.78–1.87 (m, 4H, CH2-Pyrrole), 2.63–2.66 (m, 2H, CH2-Pyrrole),
(C¼OAmide), 1655 (C¼OAmide); 1H-NMR (500 MHz, DMSO-d6): 3.49–3.52 (m, 1H, CHChiral), 3.77–3.80 (m, 1H, O-CH2-Diastropic),
1.78–1.84 (m, 4H, CH2-Pyrrole), 2.62–2.65 (m, 2H, CH2-Pyrrole), 3.97–4.01 (m, 1H, O-CH2-Diastropic), 4.58 (s, 2H, N-CH2), 7.00–7.05 (m,
3.43–3.46 (m, 1H, CHChiral), 3.73 (d, J ¼ 13.0 Hz, 1H, O-CH2-Diastropic), 3H), 7.36 (d, J ¼ 7.5 Hz, 1H), 7.41 (d, J ¼ 7.5 Hz, 1H), 7.42–7.44 (m,
4.10 (d, J ¼ 13.0 Hz, 1H, O-CH2-Diastropic), 4.58 (s, 2H, N-CH2), 1H), 7.45 (d, J ¼ 7.0 Hz, 2H), 7.48 (t, J ¼ 8.0 Hz, 1H), 8.03 (d,
6.95–7.02 (m, 3H), 7.27 (t, J ¼ 7.0 Hz, 2H), 7.35 (d, J ¼ 7.0 Hz, 2H), J ¼ 8.0 Hz, 1H), 8.07 (d, J ¼ 7.0 Hz, 1H), 8.18 (s, 1H), 8.71 (s, 1H, NH);
7.41 (d, J ¼ 7.0 Hz, 1H), 7.59 (t, J ¼ 7.0 Hz, 2H), 8.02 (d, J ¼ 8.0 Hz, 13 C-NMR (125 MHz, DMSO-d6): 22.52, 26.61, 47.25, 57.36, 62.40,
1H), 8.08 (s, 1H), 8.56 (s, 1H, NH); 13 C-NMR (125 MHz, DMSO-d6): 117.5, 117.8, 122.8, 127.2, 128.5, 129.9, 130.9 (2 C), 131.7, 131.9,
22.4, 27.6, 45.6, 57.3, 62.4, 117.5, 117.7, 119.2, 120.9, 127.2, 128.4, 135.1, 136.2, 139.9, 143.8, 144.5, 151.9, 162.4, 166.7, 179.1; Anal.
130.1, 131.4, 131.6, 135.7, 136.9, 142.4, 151.5, 160.9, 161.6, 163.5, Calcd. For C27H24N4O8S: C, 57.44; H, 4.28; N, 9.92; Found: C, 57.71;
181.2; Anal. Calcd. For C27H24FN3O6S: C, 60.33; H, 4.50; N, 7.82; H, 4.46; N, 10.17.
Found: C, 60.05; H, 4.82; N, 7.55; MS (m/z, %): 537 (Mþ, 41), 385
(28), 152 (35), 240 (49), 146 (100), 97 (26), 77 (29).
(S)-2–(2,3-Dioxo-5-((2-(phenoxymethyl)pyrrolidin-1-yl)sulpho-
nyl)indolin-1-yl)-N-(4-nitrophenyl)acetamide (20h): White solid;
(S)-N-(4-chlorophenyl)-2–(2,3-dioxo-5-((2-(phenoxymethyl)pyr- Yield: 51%; m.p. 162–164 ꢁC; IR (KBr, cmꢀ1): 1718 (C¼OKetone), 1688
rolidin-1-yl)sulphonyl)indolin-1-yl)acetamide (20d): White solid; (C¼OAmide), 1655 (C¼OAmide); 1H-NMR (500 MHz, DMSO-d6):
Yield: 48%; m.p. 209–211 ꢁC; IR (KBr, cmꢀ1): 1718 (C¼OKetone), 1700 1.78–1.82 (m, 4H, CH2-Pyrrole), 2.59–2.63 (m, 2H, CH2-Pyrrole),
(C¼OAmide), 1670 (C¼OAmide); 1H-NMR (500 MHz, DMSO-d6): 3.44–3.47 (m, 1H, CHChiral), 3.86–3.89 (m, 1H, O-CH2-Diastropic),
1.78–1.84 (m, 4H, CH2-Pyrrole), 2.61–2.63 (m, 2H, CH2-Pyrrole), 4.06–4.10 (m, 1H, O-CH2-Diastropic), 4.59 (s, 2H, N-CH2), 6.98–7.02 (m,
3.44–3.48 (m, 1H, CHChiral), 3.99 (d, J ¼ 13.0 Hz, 1H, O-CH2-Diastropic), 3H), 7.35–7.53 (m, 5H), 8.13–8.15 (m, 1H), 8.15–8.18 (m, 2H), 8.20

1682
L. FIROOZPOUR ET AL.
(s, 1H), 8.62 (s, 1H, NH); 13 C-NMR (125 MHz, DMSO-d6): 22.5, 27.0, detected using an EnVision (PerkinElmer, Wellesley, MA, USA) at
45.5, 57.1, 61.3, 117.5, 117.8, 127.2, 128.2, 129.9, 130.9, 131.7,
kex ¼ 360 nm and kem¼ 460 nm. The initial rate of hydrolysis was
131.9, 135.1, 135.7, 139.9, 142.5, 144.1, 150.7, 161.9, 166.8, 180.0;
Anal. Calcd. For C27H24N4O8S: C, 57.44; H, 4.28; N, 9.92; Found: C,
57.77; H, 4.52; N, 9.66.
determined using the early linear region of the enzymatic reaction
curve. For IC50 determination, about 8 concentrations of the syn-
thesised compounds were freshly prepared by three-fold serial
dilutions DMSO and the assay buffer such that following the add-
ition of the inhibitors, DMSO concentration would equal to 0.2%.
and GraphPad Prism 5 software was used to calculate the
IC50 values.
(S)-2–(2,3-Dioxo-5-((2-(phenoxymethyl)pyrrolidin-1-yl)sulpho-
nyl)indolin-1-yl)-N-(4-(trifluoromethoxy)phenyl)acetamide (20i):
White solid; Yield: 56%; m.p. 218–220 ꢁC; IR (KBr, cmꢀ1): 1724
1
(C¼OKetone), 1690 (C¼OAmide), 1668 (C¼OAmide); H-NMR (500 MHz,
DMSO-d6): 1.80–1.84 (m, 4H, CH2-Pyrrole), 2.60–2.68 (m, 2H, CH2-
Pyrrole), 3.70–3.73 (m, 1H, CHChiral), 3.90 (d, J ¼ 13.0 Hz, 1H, O-CH2-
Diastropic), 4.14 (d, J ¼ 13.0 Hz, 1H, O-CH2-Diastropic), 4.57 (s, 2H, N-
CH2), 7.03 (t, J ¼ 7.5 Hz, 2H), 7.32 (t, J ¼ 7.5 Hz, 1H), 7.43–7.47 (m,
3H), 7.53 (d, J ¼ 7.0 Hz, 1H), 7.59 (d, J ¼ 8.0 Hz, 2H), 7.72 (d,
J ¼ 8.0 Hz, 1H), 7.92 (d, J ¼ 7.0 Hz, 1H), 8.10 (s, 1H), 8.90 (s, 1H,
NH);13C-NMR (125 MHz, DMSO-d6): 22.5, 27.8, 44.8, 57.2, 61.4,
117.6, 119.1, 120.8, 125.3, 127.2, 127.7, 128.9, 129.3, 131.0, 133.5,
135.3, 136.2 (2 C), 144.7, 150.8, 162.0, 166.5, 179.0; Anal. Calcd. For
C28H24F3N3O7S: C, 55.72; H, 4.01; N, 6.96; Found: C, 55.89; H, 4.25;
N, 7.15; MS (m/z, %): 603 (Mþ, 60), 442 (54), 385 (29), 240 (52), 161
(42), 146 (100), 93 (17), 77 (29).
(S)-2–(2,3-Dioxo-5-((2-(phenoxymethyl)pyrrolidin-1-yl)sulpho-
nyl)indolin-1-yl)-N-(p-tolyl)acetamide (20j): White solid; Yield:
48%; m.p. 209–211 ꢁC; IR (KBr, cmꢀ1): 1723 (C¼OKetone), 1677
(C¼OAmide), 1649 (C¼OAmide); 1H-NMR (500 MHz, DMSO-d6):
1.76–1.81 (m, 4H, CH2-Pyrrole), 2.09 (s, 3H), 2.59–2.62 (m, 2H, CH2-
Pyrrole), 3.44–3.48 (m, 1H, CHChiral), 3.79 (d, J ¼ 13.0 Hz, 1H, O-CH2-
Diastropic), 4.10 (d, J ¼ 13.0 Hz, 1H, O-CH2-Diastropic), 4.51 (s, 2H, N-
CH2), 7.01–7.04 (m, 3H), 7.13 (d, J ¼ 7.0 Hz, 1H), 7.23 (d, J ¼ 7.5 Hz,
2H), 7.34 (t, J ¼ 7.5 Hz, 2H), 7.40 (d, J ¼ 7.0 Hz, 2H), 8.12 (d,
J ¼ 7.5 Hz, 1H), 8.19 (s, 1H), 8.50 (s, 1H, NH); 13 C-NMR (125 MHz,
DMSO-d6): 18.6, 24.5, 28.2, 45.5, 57.9, 61.4, 117.8, 119.2, 120.7,
126.5, 127.2, 128.2, 129.4, 130.8, 131.5, 135.1, 135.7, 137.0, 139.9,
151.5, 162.6, 166.9, 179.8; Anal. Calcd. For C28H27N3O6S: C, 63.03;
H, 5.10; N, 7.87; Found: C, 63.38; H, 5.35; N, 8.13; MS (m/z, %): 533
(Mþ, 52), 385 (42), 240 (38), 148 (100), 93 (37), 77 (49).
Computational studies
Docking procedure was performed via Autodock Tools (1.5.6). The
crystallographic structure of human caspase-3 complexed with isa-
tin sulphonamide (PDB ID: 1GFW) were retrieved from the Protein
Data Bank. The co-crystallized ligand and water molecules were
eliminated and the protein was converted to the pdbqt format
using Autodock Tools (1.5.6). Compounds structures were drawn
and 3 D-optimized using Marvin Sketch 15.8.10, 2015, ChemAxon
(http://www.chemaxon.com), then converted to pdbqt by
Autodock Tools. Each docking system were completed by 50 runs
and the grid box parameters were set as follows: size_x ¼ 50;
size_y ¼ 50; size_z ¼ 50; centred on co-ligand’s position in PDB
complex. Other parameters of Autodock search by the Lamarckian
genetic algorithm (LGA) were left as default except population
size and maximum number of evaluations which were changed to
100 and 1000000, respectively. Finally, interactions of the com-
pounds were illustrated by discovery studio visualiser ver.4.5 to
investigate their binding mode. Docking validation were con-
firmed through re-docking of 1GFW co-ligand into the receptor
with the same docking parameters of the compounds.
Disclosure statement
No potential conflict of interest was reported by the author(s).
(S)-2–(2,3-Dioxo-5-((2-(phenoxymethyl)pyrrolidin-1-yl)sulpho-
nyl)indolin-1-yl)-N-(4-methoxyphenyl)acetamide (20k): White
solid; Yield: 48%; m.p. 204–206 ꢁC; IR (KBr, cmꢀ1): 1720
(C¼OKetone), 1680 (C¼OAmide), 1656 (C¼OAmide); H-NMR (500 MHz,
Funding
1
This work was partially supported by the National Health and
Family Planning Commission of China [2012ZX09304-011,
2013ZX09401003-005, 2013ZX09507001 and 2013ZX09507-002],
Shanghai Science and Technology Development Fund
[15DZ2291600] and the Thousand Talents Program in China. This
work was also partially supported and funded by a grant from
Iran National Science Foundation (INSF); Grant no: 96011863.
DMSO-d6): 1.78–1.83 (m, 4H, CH2-Pyrrole), 2.60–2.62 (m, 2H, CH2-
Pyrrole), 3.46–3.50 (m, 1H, CHChiral), 3.62 (s, 3H), 4.14–4.21 (m, 2H, O-
CH2), 4.64 (s, 2H, N-CH2), 6.90 (d, J ¼ 7.5 Hz, 2H), 7.00–7.03 (m, 3H),
7.27–7.31 (m, 2H), 7.41 (d, J ¼ 7.8 Hz, 1H), 7.54 (d, J ¼ 7.5 Hz, 2H),
8.08 (d, J ¼ 7.8 Hz, 1H), 8.18 (s, 1H), 8.61 (s, 1H, NH); 13 C-NMR
(125 MHz, DMSO-d6): 22.5, 28.5, 46.6, 55.1, 58.3, 62.6, 109.5, 110.1,
119.3, 121.0, 127.2, 128.4, 130.2, 131.4, 131.5, 135.2, 135.8, 140.0,
142.6, 152.1, 158.5, 161.5, 163.4,179.3; Anal. Calcd. For
C28H27N3O7S: C, 61.19; H, 4.95; N, 7.65; Found: C, 61.38; H, 5.12; N,
7.82; MS (m/z, %): 549 (Mþ, 40), 385 (21), 164 (37), 146 (100), 108
(42), 77 (61).
ORCID
Massoud Amanlou
http://orcid.org/0000-0002-8559-1668
Caspase-3 and -7 inhibition assay
References
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